The present invention relates to PDGF receptor kinase inhibitory compounds and pharmaceutical compositions such as, but not limited to, slow release compositions. More particularly, the present invention relates to enriched or purified geometrical isomers of compounds of the quinoxaline family known to be PDGF receptor kinase inhibitors, compositions including same, methods of their synthesis, purification and formulation and their use for treatment of proliferative malignant and non-malignant diseases or disorders, such as, but not limited to, psoriasis, hepatic cirrhosis, diabetes, atherosclerosis, restenosis, vascular graft restenosis, in-stent stenosis, angiogenesis, ocular diseases, pulmonary fibrosis, obliterative bronchiolitis, glomerular nephritis, rheumatoid arthritis and PDGF receptor associated malignancies, such as, but not limited to, leukemias and lymphomas.
Platelet-derived growth factor (PDGF) is a potent mitogen for mesenchymal, glial, and capillary endothelial cells (for reviews, see, [1] and [2]). The three isoforms of PDGF, PDGF-AA, PDGF-AB, and PDGF-BB, interact differentially with structurally related receptors designated PDGF xcex1- and xcex2-receptors. Each of these receptors has an extracellular part featuring five immunoglobulin-like domains, a lipophilic transmembrane domain and an intracellular part with a tyrosine kinase domain containing a characteristic insert amino acid sequence [3-5]. The tyrosine kinase activity of these receptors is essential for transmission of the mitogenic signal into the cell [6].
PDGF and its receptors participate in various physiological processes such as embryonal development and wound healing. An abnormally high activity of PDGF is believed to play a central role in the etiology of certain adverse pathophysiological situations, such as atherosclerosis and restenosis [7, 8], as well as in other non-malignant diseases such as pulmonary fibrosis [9], glomerular nephritis [10], and rheumatoid arthritis [11]. Moreover, the PDGF B-chain was acquired as the sis oncogene by the acutely transforming simian sarcoma virus [12, 13]. The expression of a PDGF-like growth factor in cells infected with simian sarcoma virus or transfected with the sis oncogene leads to their transformation due to the persistent autocrine stimulation of the resident PDGF receptors.
Furthermore, certain human tumors possess PDGF receptors and express the genes for PDGF which suggest that autocrine growth stimulation via PDGF receptors contributes to the malignant phenotype of these tumors [2, 14].
The fact that PDGF is likely to be involved in the development of certain disorders has prompted the search for agents to block the action of PDGF. The approaches for interference with PDGF-induced signalling include peptides competing with PDGF for receptor binding [15], dominant negative mutants of PDGF [16, 17] or of PDGF receptor [18], and low molecular weight blockers of the receptor tyrosine kinase activity known as tyrphostins [19, PCT/US98/16232].
Certain tyrphostins which block PDGF-dependent proliferation of rabbit vascular smooth muscle cells [20] and of human bone marrow fibroblasts [21] have already been reported.
A novel class of tyrosine kinase blockers represented by the tyrphostins AG1295 and AG1296 was described by Kovalenko et al. [22]. These compounds inhibit selectively the platelet-derived growth factor (PDGF) receptor kinase and the PDGF dependent DNA synthesis in Swiss 3T3 cells and in porcine aorta endothelial cells (EC) with 50% inhibitory concentrations below 5 and 1 xcexcM, respectively. These PDGF receptor blockers have no effect on epidermal growth factor receptor autophosphorylation, weak effects on DNA synthesis stimulated by insulin, by epidermal growth factor, or by a combination of both and over an order of magnitude weaker blocking effect on fibroblast growth factor-dependent DNA synthesis.
AG1296 potently inhibits signalling of human PDGF xcex1- and xcex2-receptors as well as of the related stem cell factor receptor (c-Kit) but has no effect on autophosphorylation of the vascular endothelial growth factor receptor KDR or on DNA synthesis induced by vascular endothelial growth factor in porcine aortic endothelial cells. Treatment by AG1296 reverses the transformed phenotype of sis-transfected NIH 3T3 cells but has no effect on src-transformed NIH 3T3 cells or on the activity of the kinase p60c-src(F527) immunoprecipitated from these cells [22].
In U.S. Pat. No. 5,932,580, further low molecular weight PDGF receptor kinase inhibitors, of the quinoxaline family, are described. Specifically, substituted analogs of 1,2-dimethyl-imidazolo[5,4-g]quinoxaline were shown to selectively inhibit PDGFR autophosphorylations and proliferation of PDGFR expressing cells, like porcine arterial smooth muscle cells (SMC), porcine endothelial cells and human internal mammary artery SMC, at xcexcM concentration range.
The present invention describes enriched or purified geometrical isomers of compounds of the quinoxaline family known to be PDGF receptor kinase inhibitors, compositions including same, methods of their synthesis, purification and formulation and their use for treatment of proliferative malignant and non-malignant diseases or disorders, which show differential selectivity towards the PDGF receptor kinase. It is shown herein for the first time that geometrical isomers of compounds belonging to the quinoxaline are producable, isomerically purifyable and have differential affinity towards PDGF receptor kinase.
It is an object of the present invention to provide PDGF receptor kinase inhibitory compounds of the quinoxaline family, methods for their synthesis and purification and containment in, for example, slow release pharmaceutical compositions, and their use for treatment of a variety of diseases and disorders by local or systemic application.
According to one aspect of the present invention there is provided a preparation of a tyrphostin comprising a compound of a general formula: 
wherein,
4, 5, 6, 7, 8 and 9 indicate positions on a terminal 6-member ring;
A, B, D, X and Y are each independently selected from the group consisting of carbon, nitrogen, oxygen and sulfur;
R1, R2, R3, R5 and R7 are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, hydroxy, alkoxy, halo, C-carboxy, O-carboxy, carbonyl, thiocarbonyl, C-amido, guanyl, sulfonyl, trihalomethane-sulfonyl and a pair of electrons, or alternatively, R1 and R2 or R2 and R3 form a 5-7 member ring structure;
R6 is selected from the group consisting of alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, N-sulfonamido, S-sulfonamido, trihalomethylsulfonamido, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, C-amido, N-amido, cyano, nitro, halo, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, ureido, guanyl, guanidino, amino and a physiologically acceptable salt or a prodrug thereof;
R4 and R8 are each independently selected from the group consisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, N-sulfonamido, S-sulfonamido, trihalomethylsulfonamido, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, C-amido, N-amido, cyano, nitro, halo, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, ureido, guanyl, guanidino, amino and xe2x80x94NR10R11 and, a physiologically acceptable salt or a prodrug thereof;
R10 and R11 are each independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl and sulfonyl, or alternatively R10 and R11, form a five- or six-member heteroalicyclic ring; and, a physiologically acceptable salt or a prodrug thereof;
whereas, for Compound I, the preparation is enriched either for R6 at position 6 or for R6 at position 7, or, for Compound II, the preparation is enriched either for R6 at position 6 or for R6 at position 8.
According to further features in preferred embodiments of the invention described below, A, D, X and Y are each a nitrogen; B is a carbon; R1 and R2 are each independently selected from the group consisting of alkyl, alkoxy, halogen, nitro and amine group; R3, R5 and R7 are each a pair of electrons; R6 is an aryl, selected from the group consisting of phenyl, ferrocene, thiophene, furane, pyrrole, indole, thiazole, imidazole and pyridine.
According to still further features in the described preferred embodiments R1 and R2 are each a methyl; R4 and R8 are each a hydrogen.
According to still further features in the described preferred embodiments the preparation is enriched for Compound I in which R6 is at position 6.
According to still further features in the described preferred embodiments the preparation is enriched for Compound I in which R6 is at position 7.
According to still further features in the described preferred embodiments the preparation is enriched for Compound II in which R6 is at position 6.
According to still further features in the described preferred embodiments the preparation is enriched for Compound II in which R6 is at position 8.
According to still further features in the described preferred embodiments for Compound I, the preparation is purified either for R6 at position 6 or for R6 at position 7, or, for Compound II, the preparation is purified either for R6 at position 6 or for R6 at position 8.
According to another aspect of the present invention there is provided a pharmaceutical composition comprising, as an active ingredient, the preparation described herein and a pharmaceutically acceptable carrier.
According to further features in preferred embodiments of the invention described below, the pharmaceutically acceptable carrier is a slow release carrier.
According to still further features in the described preferred embodiments the slow release carrier is polylactic acid.
According to yet another aspect of the present invention there is provided a method of treating or preventing a protein tyrosine kinase related disorder in an organism, the method comprising the step of administering to the organism a therapeutically effective amount of the pharmaceutical composition described herein.
According to further features in preferred embodiments of the invention described below, the protein tyrosine kinase related disorder is selected from the group consisting of an EGF related disorder, a PDGF related disorder, an IGF related disorder and a met related disorder.
According to still further features in the described preferred embodiments the protein tyrosine kinase related disorder is selected from the group consisting of a cell proliferative disorder, a fibrotic disorder and a metabolic disorder.
According to still further features in the described preferred embodiments the cell proliferative disorder is selected from the group consisting of papilloma, blastoglioma, Kaposi""s sarcoma, melanoma, lung cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, astrocytoma, head cancer, neck cancer, bladder cancer, breast cancer, lung cancer, colorectal cancer, thyroid cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma, leukemia, lymphoma, Hodgkin""s disease, Burkitt""s disease, arthritis, rheumatoid arthritis, diabetic retinopathy, angiogenesis, restenosis, in-stent restenosis, vascular graft restenosis.
According to still further features in the described preferred embodiments the cell fibrotic disorder is selected from the group consisting of pulmonary fibrosis, hepatic cirrhosis, atherosclerosis, glomerulonephritis, diabetic nephropathy, thrombic microangiopathy syndromes, transplant rejection.
According to still further features in the described preferred embodiments the cell metabolic disorder is selected from the group consisting of psoriasis, diabetes, wound healing, inflammation, and neurodegenerative diseases.
According to still further features in the described preferred embodiments the organism is a mammal.
According to still further features in the described preferred embodiments the mammal is a human.
According to still another aspect of the present invention there is provided a method of locally treating or preventing a disorder of a tissue of an organism comprising the step of locally applying the pharmaceutical composition described herein onto the tissue.
According to further features in preferred embodiments of the invention described below, the tissue is selected from the group consisting of blood vessel, lung and skin.
According to an additional aspect of the present invention there is provided a method of inhibiting cell proliferation comprising the step of subjecting the cells to the tyrphostin preparation described herein.
According to further features in preferred embodiments of the invention described below, the cells are of an organism, whereas subjecting the cells to the preparation is effected in vivo or in vitro.
According to yet an additional aspect of the present invention there is provided a method of enriching a preparation of tyrphostins for a specific geometrical isomer, the method comprising the steps of (a) chromatographing the preparation through a matrix, thereby separating isomers in the preparation; (b) collecting at least one specific isomer. Optionally, the method further comprising the step of (c) crystallizing the at least one specific isomer.
According to still an additional aspect of the present invention there is provided a method for preparing a pharmaceutical composition for slow release of a tyrphostin comprising the steps of (a) providing an isomer-enriched tyrphostin preparation as described herein; (b) dissolving or dispersing a slow release carrier and the isomer-enriched tyrphostin preparation in an organic solvent for obtaining an organic solution containing the carrier and the isomer-enriched tyrphostin preparation; (c) adding the organic solution into an aqueous solution for obtaining an oil-in-water-type emulsion; and (d) evaporating the organic solvent from the oil-in-water-type emulsion for obtaining a colloidal suspension of particles containing the slow release carrier and the isomer-enriched tyrphostin preparation.
According to further features in preferred embodiments of the invention described below, the slow release carrier is polylactic acid.
According to a further aspect of the present invention there is provided a stent comprising a substantially tubular body, the body is made of a material designed for slow release of a tyrphostin preparation as described herein.
The present invention successfully addresses the shortcomings of the presently known configurations by providing new and potent tyrphostins and delivery system for treatment of a variety of disorders and diseases.